PO-390 A workflow for optimised isolation and analysis of tumour infiltrating immune subpopulations

Abstract

IntroductionSyngeneic mouse tumour models are widely used to analyse tumour immunology due to their fully competent immune-repertoire and have paved the way for novel immunotherapy agents in multiple tumour entities. However, the amount and composition of tumour infiltrating leukocytes (TIL) is highly variable. This complicates targeted analysis, in particular for small leukocyte subpopulations that may not be analysed properly or lost in the background noise. When working with large cohort sizes, immune-phenotyping by flow cytometry is time consuming and highly work intensive. We have developed improved workflows combining automated tissue dissociation with novel TIL specific isolation reagents to allow for more accurate and faster analysis of TILs and TIL subpopulations.Material and methodsWe have developed novel TIL specific enrichment reagents for the magnetic cell sorting (MACS) based isolation directly from dissociated tumour tissue. The composition of these populations before and after separation was analysed by flow cytometry. The developed tools were combined with optimised tissue dissociation and flow panels to establish comprehensive workflows.Results and discussionsWe have established workflows combining optimised and automated tissue dissociation using the gentleMACS platform with TIL specific isolation to improve and accelerate downstream analysis. Isolation of TIL was improved by developing new CD45, CD4+, CD8+, and pan T cell enrichment reagents for MACS-based isolation directly from dissociated tumour tissue. Applying this workflow, CD45 +TIL were enriched to purities above 80%–90% with high yields (>70%) from divers syngeneic mouse tumours. This allowed for rapid and reliable analysis even of small TIL subpopulations. Moreover, by using the newly developed T cell reagents, T cells were enriched to purities higher than 90%, allowing for a reliable analysis of rare T cell subpopulations.Importantly, while TIL or T cell enrichment significantly reduced analysis time and reagent costs in immune subset analysis, the composition of infiltrating cells was not affected, excluding the risk of introducing a bias by this method.ConclusionWe have developed improved workflows for the isolation of generic TIL and T cells from mouse tumours reducing time and costs of downstream analysis while standardising and enhancing the detection and quantification of immune cell subpopulations. CD45 +TILs, pan-, CD4+- or CD8+-T cells can directly be isolated from dissociated mouse tumours and analysed for subpopulations.